Analyses of the APC and TGF-β Type II Receptor Genes, and Microsatellite Instability in Mucosal Colorectal Carcinomas

APC and transforming growth factor-β type II receptor (TGF-β RII) gene mutations, and microsatcllitc instability have been found in sporadic colorectal carcinomas. To clarify further the early alterations in colorectal carcinogenesis, we investigated these genetic changes in 23 protruding- and 24 superficial-type mucosal colorectal carcinomas. TGF-β RII gene mutations and microsatellite instability were rarely found in these lesions. Nevertheless, APC was mutated in 16 of the 47 (34.0%) mucosal colorectal carcinomas and was significantly more frequently mutated in protruding- (I) and superficial elevated-type (Ila) (14/32,43.8%) than in other superficial-type (IIa+IIc, IIb, IIc, and IIc+IIa) (2/ 15,13.3%) mucosal colorectal carcinomas (P<0.04). These results indicate that the APC gene may be involved from the beginning in the tumorigenesis of many early colorectal carcinomas, particularly of the protruding and superficial elevated types. However, there might be a distinct pathway for other superficial-type colorectal carcinomas, possibly not involving APC as an initial step of tumorigenesis.     

K-ras Gene Mutation in Early Ductal Lesions Induced in a Rapid Production Model for Pancreatic Carcinomas in Syrian Hamsters

The presence of K- ras gene mutation was examined in experimentally induced preneoplastic pancreatic ductal lesions. Syrian hamsters received 70 mg/kg of N -nitrosobis(2-oxopropyl)amine (BOP) followed by repeated exposure to an augmentation pressure regimen consisting of choline-deficient diet combined with dl -ethionine and l -methionine and administration of 20 mg/kg BOP. After two augmentation pressure cycles, pancreatic ductal cell hyperplasias appeared and after three cycles, atypical hyperplasias of pancreatic ductal cells and intraductal carcinomas developed. K- ras mutations were detected by single-strand conformation polymorphism analysis of polymerase chain reaction products and nucleotide sequencing. The results showed that K- ras mutation had occurred in one of 9 simple hyperplasias of pancreatic ductal epithelium, in 5 of 9 atypical hyperplasias, and in 4 of 8 intraductal carcinomas. The findings thus suggested that K- ras is activated in association with very early stage malignant transformation of pancreatic ductal cells in hamsters.     

Higher Frequency of Point Mutations in the c-K-ras2 Gene in Human Colorectal Adenomas with Severe Atypia than in Carcinomas

Human colorectal carcinomas may be induced from adenomas or they may occur de novo. To examine which is the main pathway, we analyzed point mutations at codon 12 in the c-K- ras 2 gene in 73 colorectal carcinomas, 13 metastatic tumors, 72 adenomas and 30 normal tissues. The c-K- ras 2 codon 12 mutation frequency was 0/30 in normal tissues, 0/17 in adenomas with mild atypia, 3/37 (8.1%) in adenomas with moderate atypia, 15/18 (83.3%) in adenomas with severe atypia, 19/73 (26.0%) in primary carcinomas and 3/13 (23.1%) in metastatic tumors. The mutation frequency in adenomas with severe atypia was much higher than that in carcinomas. These results indicate that many colorectal carcinomas may not be induced through adenomas with severe atypia.     

Not infrequent K-ras mutations in depressed-type early colorectal carcinomas larger than 10 mm.

The aim of this study was to elucidate whether K-ras (codons 12 and 13) mutations occur in depressed-type early colorectal carcinomas (DECas) larger than 10 mm in size. Thirty-four cases of DECas including 27 larger than 10 mm were examined for K-ras mutations by means of microdissection, PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism), and direct sequencing. Although K-ras mutation was infrequent (1/7, 14%) in small (less than 10 mm) DECas, 16/27 (59%) and 17/27 (63%) of DECas larger than 10 mm revealed codon 12, or either codon 12 or 13 mutations, respectively. None of the evaluated pathological factors except size showed a correlation with K-ras mutation. These data indicate that although K-ras mutation could not be involved in the early stage of the progression of DECas, it might play a role at a later stage when the tumor size is over 10 mm.     

Specific codon 13 K-ras mutations are predictive of clinical outcome in colorectal cancer patients, whereas codon 12 K-ras mutations are associated with mucinous histotype.

BACKGROUND: K-ras mutations, one of the earliest events observed in colorectal carcinogenesis, are mostly found in codons 12 and 13, and less frequently in codon 61, all three of which are estimated to be critical for the biological activity of the protein. Nevertheless the prognostic significance of such mutations remains controversial. Our purpose was to assess whether any or specific K-ras mutations in primary colorectal cancer had prognostic significance and were linked to clinico-pathological parameters. PATIENTS AND METHODS: Paired tumor and normal tissue samples from a consecutive series of 160 untreated patients (median of follow up 71 months), undergoing resective surgery for primary colorectal carcinoma, were prospectively studied for K-ras mutations by PCR/single strand conformation polymorphism sequencing. RESULTS: Seventy-four of the 160 (46%) primary colorectal carcinomas presented mutations in K-ras: 54% in codon 12, 42% in codon 13 (particularly G-->A transition) and 4% in both. Codon 12 K-ras mutations were associated with mucinous histotype (P <0.01), while codon 13 K-ras mutations were associated with advanced Dukes' stage (P <0.05), lymph-node metastasis (P <0.05) and high S-phase fraction (P <0.05). Multivariate analysis showed that codon 13 K-ras mutations, but not any mutation, were independently related to risk of relapse or death. CONCLUSIONS: Our results suggest that codon 12 K-ras mutations may have a role in the mucinous differentiation pathway, while codon 13 mutations have biological relevance in terms of colorectal cancer clinical outcome.     

Somatic Mutations of K-Ras and BRAF in Thai Colorectal Cancer and their Prognostic Value

Background: The study aimed to determine the incidence of K-ras and BRAF mutations in colorectal cancers(CRCs) in Thai patients and evaluate association with clinicopathological parameters including treatmentoutcomes in terms of event free survival (EFS). Materials and Methods: Two-hundred colorectal cancer specimenswere collected for studies of K-Ras codon 12, 13 and 61, and BRAF codon 600 by polymerase chain reaction anddirect nucleotide sequencing. Results: The overall incidence of K-Ras mutations in our patients was 23%. K-rasmutation frequencies in CRC stages (AJCC) I, II, III and IV were 6.7%, 16.1%, 23.3% and 26.6%, respectively(p-value>0.05). The three most common mutation forms were G12D, G12V and G13D. K-Ras mutation status wasassociated with poorer EFS in stage I-III CRCs (p-value 0.03). Conclusions: The study found a lower mutationfrequency of K-Ras and BRAF compared to reports involving other ethnic groups. However, K-Ras mutationsdid have a negative prognostic value in early-stage CRCs.     

Lower Incidence of K-ras Codon 12 Mutation in Flat Colorectal Adenomas than in Polypoid Adenomas

In order to clarify genetic changes in flat adenomas, K- ras codon 12 point mutations were examined in 56 flat adenomas, 81 polypoid adenomas and 42 cancers of colon and rectum. The mutation frequency in flat adenomas was 23% (13/56), significantly lower than that in polypoid adenomas (67%: 54/81) and cancers (76%: 32/42). Even mildly dysplastic adenomas or small (less than 5 mm) adenomas showed higher mutation incidence in polypoid type (62%, 57%) than in flat type (23%, 19%). Among flat adenomas, flat elevated lesions exhibited relatively higher mutation frequency than completely flat or depressed ones. As for cancers, 14 tumors (33%) contained mutations only in a minor tumor cell population, indicating that these mutations occur at a late stage of tumorigenesis. These results suggest that the adenoma-carcinoma sequence through flat adenomas may he different from that through polypoid adenomas, and genetic changes may be heterogeneous in colorectal carcinogenesis.     

结直肠癌内镜活检组织p53基因变异的PCR—SSCP分析

目的 探讨结直肠癌ｐ５３基因变异情况及其意义。方法 采用ＰＣＲ技术扩增内镜下活检的结直肠癌组织中ｐ５３基因第５～９外显子，再以银染色单链构象多态性（ＳＳＣＰ）技术分析其变异情况。结果 ２０例结直肠癌组织ｐ５３基因的ＰＣＲ扩增均获成功；银染色ＳＳＣＰ分析发现其中５例有异常条带，阳性率为２５．０％，主要见于第５、７外显子，第９外显子未见阳性带；３例升结直肠癌中有２例阳性。结论 结直肠癌ｐ５３基因变异率     

c-erbB-2、ras、p53基因产物在大肠癌及癌前病变中的表达

目的：探讨c－erbB－2、ras、p53基因的表达与大肠癌发生发展的关系以及其对大肠癌早期诊断、预后判断的价值。方法：对45例大肠癌及36例癌旁非腺瘤型不典型增生、17例大肠腺瘤，用免疫组化方法检测基因产物的表达。结果：p53在大肠癌的阳性表达率为57．8％，p53蛋白高表达的癌旁非腺瘤型不典型增生及大肠腺瘤均为中度或高度不典型增生。p53蛋白表达与大肠癌的组织分化程度、淋巴结转移有关（P＜0．05）。p21、p185蛋白表达与大肠癌组织学分型、癌组织浸润程度、淋巴结转移无关，而与癌组织分化程度有关（P＜0．05）。结论：p53蛋白表达可能是大肠病变恶性倾向的一个独立标志。p53、p21、p185蛋白对大肠癌的发生、发展起重要作用。     

散发性结直肠癌D10S1265位点的杂合缺失分析

目的:染色体上某特定位点遗传物质的丢失是肿瘤发生中的常见现象,抑癌基因的杂合缺失是结直肠癌形成中的关键步骤之一.本实验通过对83例散发性结直肠癌中D10S1265位点杂合缺失的研究,探讨其在结直肠癌演变中的作用.方法:荧光标记的多态性微卫星引物D10S1265与83例结直肠癌的肿瘤和正常组织进行PCR反应.PCR产物在ABI Prism 377自动荧光测序仪电泳3h,以GeneScan3.1和Genotyper 2.1软件进行扫描以及杂合缺失分析.其结果与临床病理因素进行卡方检验.结果:D10S1265位点(10q24.3)的杂合缺失率是50.0%,肝转移的7例未发现LOH,无肝转移病例达58.97%(23/39,P=0.014),但与淋巴结转移无关.另外,此位点的杂合缺失与Dukes'分期显著相关(P=0.013),与其他临床病理因素无关.结论:D10S1265位点附近可能存在与散发性结直肠癌有关的抑癌基因,此基因与结直肠癌的进展和肝转移相关.     

Significance of HPV Infection and Genic Mutation of APC and K-ras in Patients with Rectal Cancer

Background: Significance of HPV infection and genic mutation of APC and K-ras in rectal cancer has beeninvestigated but not clarified. The objective of our study was to investigate these parameters in patients with rectalcancer to analyze correlations with biological behaviour, to determine relationships among the three, and alsoto demonstrate survival prognosis effects. Methods: From December 2007 to September 2008, 75 rectal cancercases confirmed by histopathology in the Tumor Hospital of Xinjiang Medical University were enrolled. Thecontrol group consisted of normal rectal mucous membrane taken simultaneously, a least 10 cm distant from thecarcinoma fringe. HPV DNA, the MCR of APC and exon-1 of K-ras were detected by PCR and PCR-SSCP. Allresults were analyzed in relation to clinical pathological material, using chi-square and correlation analysis viaSPSS.13 and Fisher’s Exact Probability via STATA. 9.0. All 75 patients were followed up for survival analysisusing Kaplan-Meier and Log-rank tests. Results: 55 out of 75 cases demonstrated gene HPV L1 while it wasnotdetected in normal rectal mucosa tissue. HPV infection was correlated with age and lymphatic metastasis(P<0.05) but not other characteristics, such as ethnicity, tumor size, histological type, tumor type, Duke’s stageand infiltration depth. Some 43 cases exhibited APC genic mutation (57.3%) and 34 K-ras genic mutation(45.3%). APC genic mutation was correlated with gender( P<0.05), but not age, histological type, infiltrationdepth, lymphatic metastasis and Duke’s stage. In 55 cases of rectal cancer with HPV infection, there were 31cases with genic mutation of APC (56.4%) and 24 with genic mutation of K-ras (43.6%). For the 20 cases ofrectal cancer with non-HPV infection, the figures were 12 cases (60%) and 10 (50.0%), respectively, with nosignificant relation. Survival analysis showed no statistical significance for K-ras genic mutation, APC genicmutation or HPV infection (P>0.05). However, the survival time of the patients with HPV infection was a littleshorter than in cases without HPV infection. Conclusions: Our results suggest that HPV infection might be animportant factor to bring about malignant phenotype of rectal cancer and influence prognosis. Genic mutationof APC and K-ras might be common early molecular events of rectal cancer, but without prognostic effects onmedium-term or early stage patients with rectal cancer.     

Tyrosine 1045 Codon Mutations in Exon 27 of EGFR are Infrequent in Oral Squamous Cell Carcinomas

Background: The activation and inactivation of receptor tyrosine kinases are tightly regulated to ensure faithfulreplication of cells. After having transduced extracellular growth activating signals, activated EGFR is subjectedto downregulation either by clathrin mediated endocytosis or c-Cbl mediated proteasome degradation dependingon the ligand concentration. c-Cbl is an ubiquitin ligase which requires a phosphorylated tyrosine residue atposition 1045 in the cytoplasmic domain of EGFR to interact and add ubiquitin molecules. While activatingmutations in exons 19 and 21 have been associated with the development of several cancers, the status of mutationsat tyrosine 1045 coding exon 27 of EGFR remain to be investigated. Consistently, defective phosphorylation at1045 has been associated with sustained phosphorylation of EGFR in non-small lung carcinomas. Hence in thepresent study we investigated the genetic status of the tyrosine 1045 coding site within exon 27 of EGFR geneto explore for possible occurrence of mutations in this region, especially since no studies have addressed thisissue so far. Materials and Methods: Tumor chromosomal DNA isolated from thirty five surgically excised oralsquamous cell carcinoma tissues was subjected to PCR amplification with intronic primers flanking the tyrosine1045 coding exon 27 of EGFR gene. The PCR amplicons were subsequently subjected to direct sequencing toelucidate the mutation status. Results: Sequence analysis identified no mutations in the tyrosine 1045 codon ofEGFR in any of the thirty five samples that were analyzed. Conclusions: The lack of identification of mutation inthe tyrosine 1045 codon of EGFR suggests that mutations in this region may be relatively rare in oral squamouscell carcinomas. To the best of our knowledge, this study is the first to have explored the genetic status of exon27 of EGFR in oral squamous cell carcinoma tissue samples.     

Frequent Loss of Heterozygosity at the MCC Locus on Chromosome 5q21-22 in Sporadic Colorectal Carcinomas

Recent studies have identified a gene on chromosome 5q, designated MCC (mutated in colorectal cancers), as a candidate for the putative colorectal tumor suppressor gene that is located at 5q21. We examined loss of heterozygosity (LOH) at the MCC locus and its vicinity in sporadic colorectal carcinomas, using 12 RFLP (restriction fragment length polymorphism) markers. One clone, L5.71, had been used to identify the MCC gene; all 12 markers also had tight linkage to the gene responsible for adenomatous polyposis coli. All 40 cases studied were informative with at least one marker, and 22 of them (55%) showed LOH at one or more loci. LOH in the tumors was more frequent in the immediate vicinity of L5.71 than in distant parts of the chromosome, and a common region of deletion was detected between markers L5.62 and 15A6. In one case, alleles were retained at L5.71 and at loci proximal to L5.71, but alleles were lost at loci distal to L5.71. In another case, both alleles were retained at L5.71 but alleles were lost at loci proximal and distal to L5.71. These results support the conclusion that a tumor suppressor gene for colorectal carcinoma is located within or around locus L5.71.     

血清糖链抗原、粘附分子及K-ras基因突变水平在胰腺癌中的表达及其临床意义

目的 探讨血清糖链抗原、粘附分子及K-ras基因突变联合检测在胰腺癌诊断中的应用价值,及其在胰腺癌中的表达意义.方法 收集109例胰腺癌患者(Ⅰ期14例、Ⅱ期33例、Ⅲ期39例、Ⅳ期23例;淋巴结转移56例,无淋巴结转移53例)、78例胰腺炎患者和98例健康体检者血清.采用化学发光法检测血清CA50、CA125、CA199、CA242水平,采用双抗夹心法酶联免疫吸附试验法检测ICAM、VCAM、ALCAM、CEACAM水平,采用聚合酶链反应法检测K-ras基因突变情况.结果 胰腺癌组CA50、CA125、CA199、CA242、VCAM、ALCAM、CEACAM水平和K-ras突变率明显高于胰腺炎组和健康对照组(P＜0.05);胰腺炎组VCAM、ALCAM、CEACAM水平明显高于健康对照组(P＜0.05),而胰腺炎组和健康对照组CA50、CA125、CA199、CA242水平和K-ras突变率比较,差异无统计学意义(P＞0.05).联合检测[logit (P)=0.114×CA199+0.235×CEACAM+ 0.082×K-ras]对胰腺癌的诊断价值最大,AUC达0.931,显著高于各个指标单独检测,其敏感性和特异性分别达94.5％和86.9％.VCAM、ALCAM、CEACAM水平随胰腺癌分期的增加,水平不断升高(P＜0.05);有淋巴结转移的胰腺癌患者粘附分子VCAM、ALCAM、CEACAM水平均明显高于无淋巴结转移胰腺癌患者(P＜0.05).结论 糖链抗原、粘附分子和K-ras基因突变联合检测能够大大提高胰腺癌的诊断效能,其中粘附分子表达水平与胰腺癌的分期和淋巴结转移密切相关.     

Detection of K-ras Point Mutations in Mesenteric Venous Blood from Colorectal Cancer Patients by Enriched Polymerase Chain Reaction and Single-strand Conformation Polymorphism Analysis

In order to confirm the presence of cancer cells in mesentericvenous blood and to examine their relationship with the occurrenceof liver metastases, we attempted to detect K-ras codon 12 pointmutations in perioperative mesenteric blood using enriched polymerasechain reaction and single-strand conformation polymorphism (PCR-SSCP)analysis in 25 patients with primary colorectal tumors carryingK-ras point mutations. Among these patients, three with synchronousliver metastases were included. The same K-ras point mutation(substitution of GAT for GGT) was detected in both the bloodand the primary tumor in a Dukes' C patient. We confirmed thisresult by colony hybridization and estimated the tumor-to-normalcell ratio to be 1:400. This patient has no liver metastasestwo years after surgery and her carcinoembryonic antigen (CEA)level remains normal. We demonstrated that considerable numbersof cancer cells can be found in mesenteric venous blood duringcolorectal cancer surgery. However, their potential role inthe formation of liver metastases remains unclear.     

Study on the Expression and Significance of TGF-β1, p-ERK1/2and K-ras in Colorectal Cancer Using Tissue Microarray Technique

OBJECTIVE This study aimed to explore the expression and significance of transforming growth factor β1(TGF-β1),extracellular signal-regulated kinases 1/2 (ERK1/2), and K-ras in colorectal cancer (CRC) using tissue microarray technology.METHODS The expressions of TGF-β1, ERK1/2, and K-ras in colon cancer cells taken from the specimens of 92 CRC patients (stage Ⅰ: 16 cases, stage Ⅱ: 28 cases, stage Ⅲ: 24 cases, and stage Ⅳ:24 cases) were analyzed using tissue microarray technology and immunohistochemistry, and compared with those of 20 normal colon tissue samples.RESULTS High immunoreactive scores (IRS) of TGF-β1,p-ERK1/2, and K-ras protein in CRC were obtained, which were 66.3% (61/92), 59.8% (55/92), and 48.9% (45/92), respectively, and those in normal epithelial cells of colon were 10% (2/20), 20% (4/20), and 30% (6/20), respectively (P < 0.05). The expressions of TGF-β1 and ERK1/2 in CRC at stage Ⅰwere 37.5% and 31.3%,respectively, and those in CRC at stage Ⅳ were 83.3% and79.3%, respectively, with statistically significant differences. No significant relationship was found between K-ras expression and tumor stages (P>0.05).CONCLUSION High level expressions of TGF-β1 and ERK1/2 are closely related to the clinical stages of colon cancer and crosstalk may exist between the 2 signal pathways.     

结肠癌p16基因缺失与突变的研究

目的 :探讨p16抑癌基因外显子 2缺失、突变与结肠癌发生、发展的关系。方法 :采用聚合酶链反应 (PCR)、聚合酶链反应—单链构象多态性 (PCR SSCP)分析方法检测结肠癌中p16基因外显子 2的缺失、突变。结果 :30例结肠癌样本中 ,高分化及低分化腺癌各 1例PCR扩增无扩增产物 ,可能为p16基因缺失 ;其余 2 8例结肠癌、正常组织均有产物出现。SSCP分析 ,30例结肠癌中未见异常泳动带 ,无p16基因突变。结论 :p16基因外显子 2缺失可能很少参与结肠癌的发生发展 ;而p16基因突变可能与结肠癌发生无关。     

Microsatellite Instability and Frameshift Mutations in the Bax Gene in Hereditary Nonpolyposis Colorectal Carcinoma

We studied microsatellite instability (MI) and bax gene abnormalities in colorectal carcinomas from 36 patients diagnosed as having hereditary nonpolyposis colorectal cancers (HNPCC) according to the clinical criteria (12 with confirmed HNPCC in group A and 24 at high risk of HNPCC in group B) and from 20 randomly selected patients with other colorectal cancers. MI was examined at 4 dinucleotide microsatellite loci and one mononucleotide locus. Frameshift mutations in the bax gene were detected by polymerase chain reaction-single strand conformation polymorphism analysis. MI was detected in 7 of the 12 patients in group A and 12 of the 24 in group B. Three MI patterns were identified: type 1, MI in both mono- and dinucleotide repeats; type 2, MI only in mononucleotide repeats and type 3, MI only in dinucleotide repeats. Most MI-positive patients in group A showed type 1 MI, whereas in group B, 5 showed type 1, 3 showed type 2 and 4 showed type 3. Frameshift mutations in the bax gene correlated strongly with type 1 and type 2 MI. These results indicate that mutations of different DNA mismatch repair genes may cause several types of MI and result in several different clinical phenotypes of HNPCC. The bax gene may be one of the target genes which play a role in the tumorigenesis of HNPCC.     

静脉联合腹腔化疗治疗晚期胃肠肿瘤87例临床分析

目的　评价静脉联合腹腔化疗治疗对晚期胃肠肿瘤治疗效果和副作用。方法　对 1996年 3月至 2 0 0 0年 11月间 ,我科对采用吡喃阿霉素静推、羟基喜树碱静滴、5 -FU及顺铂腹腔灌注方法治疗 87例晚期胃肠肿瘤进行分析 ,总结其近期治疗效果。结果　本组 87例 ,总有效率为 5 2 9% (4 6/87) ,中位生存期为 15 7月 ,且毒副反应较轻。结论　静脉联合腹腔化疗是治疗晚期胃肠肿瘤的有效治疗手段 ,副反应轻 ,病人易于接受     

PCR-SSCP检测大肠癌组织中p53、K-ras基因的突变

目的：探讨p53、K-ras基因突变与大肠癌发生、发展的关系。方法：应用PCR－SSCP方法检测68例大肠癌和癌旁组织以及20例正常组织中p53、K-ras基因突变情况。结果：大肠癌组织中p53、K-ras基因突变率分别为47．1％（32／68）和44．1％（30／68），明显高于癌旁组织（13．2％，9／68；7．4％，5／68），20例正常组织中未检出p53、K-ras基因突变。伴有淋巴结及远处转移的大肠癌者，其p53、K-ras基因突变率明显高于无淋巴结及远处转移者；p53、K-ras基因突变与大肠癌组织学类型无关。结论：p53、K-ras基因突变与大肠癌发生、发展有密切关系，其在细胞癌变中起重要作用，可作为评估大肠癌转移的分子生物学指标之一。